ABSTRACT: Various disciplines have emerged due to vast expansion in the studies of plant sciences. Discovery of new methods, protocols and analytical techniques facilitates for thorough and deep study of the given subject. One among many disciplines which has appeared as an offshoot from basic plant science is forensic botany. Forensic botany is an interdisciplinary area where the knowledge of botany is applied to solve the crime. This paper introduces about the basic of the subject explaining various sub-disciplines and its application in forensic science. It also cites some cases where botanical evidences have been the part of judgements.

Plants since the beginning of human existence have played important role in their civilizations. They have been used as both weapons and protector. The role of plants as evidence for solving the criminal cases though is comparatively new. It was only after 1932 when during the trial of Lindbergh kidnapping case the botanical evidences were accepted (Graham, 1997) and the judgement was made based on them the plants were recognised as crime solvers. This case marked the birth of Forensic Botany.

The term Forensic comes from the Latin word ‘forensis’ meaning “of or for the forum”. Forensic Science is often defined as the application of scientific methods and techniques for the purposes of justice. Forensic Science has been branched into various headings viz. forensic biology, forensic anthropology, forensic chemistry, forensic physics, forensic botany, forensic entomology, forensic serology, etc.

Forensic Botany is defined as the use of plant evidence in court. In other words, examination of plant materials or remains collected at a crime scene and using them to solve crimes or other legal problems may be loosely termed as Forensic Botany. Forensic botany can play a valuable role in criminal investigations, but it has been underused because of the lack of botanical knowledge among most people involved in criminal investigations. Although forensic botany has been underused in criminal investigations over the years, resourceful investigators and scientists with initiative are beginning to change this.

Forensic Botany is subdivided into several specialities of botanical fields like, plant taxonomy, palynology, plant ecology, limnology etc. and more recently molecular plant biology. Forensic botany is useful in the examination of gastric contents in homicide victims. It can help determine the contents and location of a victim's last meal. If a victim has been moved from the location of death, plant analysis can determine whether plant remnants on the body are characteristic of the vegetation where the body was found or of some other location where the victim was killed. Finding of clandestine graves requires the expertise of plant ecologists who can recognize localized, unusual areas of plant succession.

This paper is an attempt by the authors to discuss different sub-disciplines of forensic botany with some suitable illustrations. Also the authors have attempted to present a few interesting cases in which botanical evidence have been the part of judgements.

MAJOR SUBDISCIPLINES OF FORENSIC BOTANY

Ecology and Limnology

Ecology is the branch of biology that deals with the relations of organisms to one another and to their physical surroundings. When we take plant ecology, it includes the distribution abundance of plants in a region, their effects upon environmental factor and interactions among and between plants and other organisms. Knowledge of ecology is useful for the forensic investigators in determining the relation of victims, crime scene and suspects. As a forensic ecologist one need to know several things in relation to ecology. Not, only the structure of plants but also their natural and semi natural habitat, biotic and a-biotic component of the habitat, how one species affect others, their possible and probable time of abundance, growth, structure and overall functions. Apart from these, forensic ecologists are expected to know about garden and gardening too. They are required to keep the knowledge of plantation parks, ponds, canals, road-sides verges hedge rows and wastelands. Though forensic ecologists cannot be an expert in every aspect of ecological sciences, knowledge of vegetation, soil sediments, aquatic and terrestrial habitats are some essential areas for investigations (Whiltshire, 2009). Individual may develop an interest and subsequent expertise in any specific area. The study of the structural and functional interrelationships of organisms of inland waters as they are affected by their dynamic physical, chemical and biotic environments is called limnology. Though limnology involves the study of both freshwater and saline inland water, freshwater bodies are of more importance to human activities (Wetzel, 2001). Thus forensic limnology is more concern to freshwater ecology.

Various cases exemplify the applicability of plant ecology to forensic investigation. In one case, victim was brutally attacked to death and was drowned into a nearby pond. Fortunately the accompanied person with victim managed to escape and informed to the immediate possible option. Investigators resumed water soaked, mud encrusted footwear and clothing from the apprehended suspect. Investigators also obtained samples of pond water sediments and aquatic vegetation from the pond aiming to establish the linkage between the clothing of suspects, victims and pond biota. Investigators with the basic ecological application and certain liminological tests established the linkage. Numerous species of diatoms and planktonic algae were recovered from the victims and suspects clothing and reference samples of pond sediments (Siver et al., 1994).

Horton et al., (2006) developed a quantitative diatom based reconstruction technique to confirm drowning as cause of death and localise the site of drowning. They collected samples from the local/regional water bodies to act as control in the examination of diatom assemblages associated with lungs and clothing of the subject.

Palynology

Palynology is the branch of botany that deals with the study of pollen grains, their shape, size, structure, origin, etc. Forensic palynology involves the solving of criminal cases with the help of identification of pollen grains and spores. Pollen grains being smaller in size are not seen by the culprit and thus remain at the crime scene helping the investigator to solve the crime. Pollen grains also help in fixing the location of the crime as certain plants are found in particular regions and identification of their pollens and spores determine the location of the crime occurred. Pollen grains are microscopic and not visually obvious trace evidence during crime scene collection but are retained on clothing, embedded in carpets, pervasive in soil, etc. Pollen grain morphology can be used to identify a plant genus and often the species.

Crime scenes which are restricted to a smaller, such as a rape scene, are good choices for pollen evidence. Generally areas have a specific pollen distribution pattern representing the combination of plant species found in the surrounding vegetation. Forensically, the pollen grains which are commonly found hold less evidentiary value than those which are uncommon, or belong to the species which is poorly distributed.

In a case reported in Auckland, New Zealand, a woman alleged that the suspect had raped her in an alleyway few meters away from his car. While the suspect claimed that he never moved away from his car and nor did he entered the mentioned alleyway. Adding to this he strongly pointed out that he had never had any sexual intercourse with the victim and the soil found on his clothes is from the driveway area. During the investigation the officials collected the soil samples from both the mentioned areas and also from the suspects’ clothes. The analysis of soil samples which also contains pollen grains proved the sexual assault taking place in alleyway, the suspect was convicted later on the basis of this evidence.

Anatomy

The construction of plant’s body and its components can provide a wide variety of forensic evidences. The rigid external cell wall covering the plant cells, unlike the animal cells, does not allow the plant cells to wash away or degenerate easily. The varying percentage of cellulose and lignin making up the cell wall are not digestible by animals except ruminants, termites and a few other anthropods as far as cellulose is concerned whereas with lignin it is somewhat more difficult to digest due to its being more complex structurally and chemically which can be broken down only by a few fungi. In addition to this, cells that make up wood have a secondary wall that has much higher percentage of lignin, which adds on to the strength and long life of these cells, proof of which stands tall with the wooden churches of Norway which were built during the end of the eleventh century and the still remain standing and are in use.

In forensic science, the identification of wood is done on the basis of the physical and anatomical properties of wood. Physical properties include colour, odour, weight, hardness, lustre and texture of the wood sample. Also, the water extract of some timbers show distinct fluorescence which helps in identifying the unknown wood samples. Anatomical properties include whether the pores are present or absent and if present then the further identification s done on determining the pore number, pore size and pore arrangement.

The manner in which cell walls are laid down and the patterns formed by cells are peculiar to given taxonomic groups. The herbaceous (non woody) plants have characteristic cell shapes, sculpturing of the cell walls, arrangements of cells of the epidermis, and combinations of cell types in their pulpy parts. Some cells contain crystals of calcium oxalate or starch grains of types that vary among species. Identification of starch grains is based on three characteristics – size, shape, and presence or absence as well as shape of markings on the starch grains called the hilum. Identification of starch grain is easy on freshly prepared starch. In old starch or boiled starch or chemically modified starch the hilum change from dot to transverse or transverse to satellite and satellite to ruptured grain. Thus, too much reliance should not be placed on hilum unless history of starch is known. Comparison of unknown sample with known sample is always desirable. In case of decomposed starches identification should be based on few grains which are left intact.

In forensic work, plant parts specifically adapted for dispersal can be particularly useful. As plant species are each best suited to a particular set of environmental conditions, not all species grow everywhere. Plant species occur in association, or communities, whereas the environmental requirements of several species overlap. Anatomical knowledge about species can be useful forensically, because a botanist can identify plant fragments found on the clothing of a victim, and can determine whether those plants came from the site in which the victim was found or from some other area.

Correct identification of plant samples depends on trained botanists’ abilities to gain access to recorded information on the characteristics of the species to which the sample belongs. Therefore, anyone who identifies a new sample of plants must publish the contents as all information is needed and is can help in solving the criminal cases. Lancia & Conforti (2013) studied the growth rate of the bryophyte Leptodyctium riparium, was used in estimating the Post Mortem Interval of some human skeletal remains that were found in a wooded area near Perugia, in Central Italy. Forensic Plant Biotechnology

Plant Biotechnology in recent few decades has emerged as an exciting area of plant sciences. Through this technology various biological manipulation and interpretation can be done (Chawla, 2002). Techniques like DNA fingerprinting, DNA barcoding, Molecular markers are extensively used these days by investigators to solve the cases. In forensic botany it is mainly used for species identification, endemism of the subject plant.

During the course of plant species identification in the field for general academic purposes one gathers maximum morphological character in the field note book to identify the plant up-to species level. Unlike the conventional taxonomic identification the morphological character is limited in forensic investigation, plant species found as evidence material are in fragment and in such instances identification of the species with the convention available key in any flora becomes difficult. Though DNA barcoding is one option but it has limited scope in plant science as there is no gene in the plant which can be the barcode unlike in animal where the mitochondrial cytochrome c oxidase I has become universal barcode. In these circumstances one has to depend on the biotechnological tools such as ‘Short Tandem Repeats (STRs)’, ‘Single Nucleotide Polymorphisms (SNPs), ‘Simple Sequence Repeats (SSRs), Variable Number Tandem Repeats (VNTRs) (Zaya & Mary, 2012) . Discussing details of these techniques may be out of scope of this article. One can find more about techniques in ‘Plant DNA Fingerprinting and Barcoding; Methods and Protocols. Methods in Molecular Biology’ edited by Sucher et.al., (2012).

Eurlings et.al., (2013) has reported forensic identification of Rauvolfia sp. using DNA bar coding technique which can be used to control illegal international trade as manually the root samples cannot be identified at species level. Gilmore et.al., (2003) diagnosed profile of 93 cannabis plants (Cannabis sativa) using STRs. Bryophytes can be wise choice for forensic investigation in a case where the criminals walk in a forest or semi urban area and bryophytes fragments get attached to shoes even after a wearer walks for several hours. Also DNA of bryophytes stays intact in fluctuating environment which allows DNA profiling for evidence (Virtanen et. al., 2007).

Hence one can apply various tools of biotechnology in multidimensional forensic analysis and evidence.

Bioinformatics and Forensic Botany

From the above it is evident that the DNA profiling is the centre for biotechnological application in forensic botany. Forensic DNA and bioinformatics are inherently interdisciplinary and draw their techniques from statistics and computer science bringing them to bears on problems in biology and law.

Identification of plants and micro-floras reported in the crime scene can be re-sequenced using microarray and can be analysed using bioinformatics standard techniques. During the investigation objective is to match DNA recovered from crime scene and criminals. Bioinformatics tools and available database can be used to estimate and determine the probability that a particular genotype might occur (Bianchi and Lio, 2007). Plant Expression Database, PlantGDB, Gramene, PGF-DB (Plant Gene Family Database), Mendel, GreenPhyIDB, GARNet are few plant genetic databases which provides many algorithmic tools that allows users to analyze sequence data using broad array of information tools.

When the crime has taken place in grassland vegetation, grasses are preferred to as trace evidences to solve the case. Database ‘Gramene’ (www.gramene.org) may be used for a comparative analysis. It is a comparative genome mapping database for grasses. Though mainly for rice, it uses rice as a framework genome to organise information for other grass species (Doreen et.al., 2002). Botanists at the Australian National University, Canberra developed a molecular identification database for the identification of hundred Australian grasses. The identification system is built on a phylo-genetically sound framework and consists a series of taxon specific PCR assays for the progressive identification of grass samples and thus claiming it as a novel technology for forensic botany (Ward et.al., 2005, 2009).

Although the bioinformatics tools and application are more focussed toward the important crop plants, gradually it is attaining the attention of forensic botany as well. A larger database for wild grass needs to be developed and all the regional databases require to be integrated with national and international databases keeping forensic analysis in mind. Plants occurred in the past investigation should be listed out and depending upon the frequencies and complexity order research project may be formulated under the monitoring of forensic department and collaboration with research institutes and universities.

FORENSIC BOTANY AS PART OF JUDGEMENT

Forensic botany has reported a number of cases in which it was difficult to establish the correct manner of crime that has taken place. Such cases are being reported all over the world a few of them are mentioned below.

1. Investigation: In case of Ch. Hymavathi Vs Govt. Of Andhra Pradesh and others, forensic report of the poisonous substance (plant extract) used in the murder solved to identify the real culprit. Ignorance of forensic report leads to harassment of innocent acquitting the real culprit. Later the forensic report was taken into cognizance to solve the case (http://indiankanoon.org/doc/723998/).

2. Homicide or Suicide: These cases were reported in Taiwan which shows the simple use of plant material evidence to establish whether the body found is the result of homicide or suicide.

a) The first case, a body of a young woman was found in the gutter. Since no bone fractures were seen (before the autopsy) it was taken as a hit and run case. On viewing the surveillance tapes she was seen but as soon as a truck passed her she was not seen so it was concluded that she has been hit by the truck and to hide the accident the driver has hid her body in the gutter. While during the autopsy some plant material, a tiny berry and stem, were found in her hairs which were uncommon for that area suspected to be from the family Solanaceae. Later on, while the investigators visited the scene of crime they found the similar plants on the railing of the building present there whose height was such that no person walking on street can come into its contact. Thus they concluded that the woman has felled from the top of building and while falling down her body made the contact with the plants and so the plant materials were transferred to her hairs and felled into the gutter. The autopsy report mentioned the cause of death as the impact injuries and the interviews held with her relatives confirmed her suicidal tendency due to her suffering from depression. Thus, confirming the theory of investigators.

b) In another case, a male body was found hanging on a tree. The mode of the death was established as asphyxiation due to hanging but it was difficult to determine whether it was homicide or suicide. During the investigation it was found that moss has been smeared on the inside of his wrists and the identical moss was fond growing on the tree. It was concluded that the moss must have been transferred onto the skin while tying the rope to an upper branch. Thus, the manner of death was established as suicide.

3. Primary and Secondary Crime Scenes: A dead body of a young man was found in the gutter on the side of the road in Taiwan. The dead man was grasping plant material in his hand. Most of the injuries were below the knees and the autopsy report revealed the presence of fragments of bamboo in his stomach contents. But, in the gutter where the body was found no bamboo was growing. Thus, the bamboo evidence helped in locating the scene where the man was injured, it was thought that the man before dying survived on bamboo and water. The holding of grass in his hands proved that he was alive when he was in gutter. Later it was found that it was a hit and run case and guilty persons threw him in the bamboo grave to hide the accident.

4. Plant Succession and Clandestine Graves: Whenever an area covered with plants is cleared say for a purpose of grave site, then due to plant succession process after sometime plants re-grow there. By analysing the soil conditions and plant growth one can estimate about the time of death at such sites. An example of this is the post mortem dating of a skeleton found in a field by determining the age of the plant growing in the eye socket of the skull.

5. Time of Death: One of the cases reported in Colorado, marks as an example for determining the time of death by analysing the stomach contents. A man was suspected to be murdered by his own wife, Jil Caroll, he was her eighth husband. During investigation was found that the deceased was in habit of having the same breakfast daily. During autopsy, the stomach contents were misidentified as noodles later on they were reported as potatoes. The knowledge of estimated time to digest this type of food, it was concluded the he died between two to four hours after the breakfast which was not consistent with the wife’s alibi. Later she was convicted for the murder.

CONCLUSION

Forensic botany, though, not new but has emerged as an important discipline in solving the mysteries and crimes. It is an interdisciplinary of various branches of botany where a forensic botanist has to acquire knowledge of different fields of botany. With recent advancement in molecular technology investigation with application of forensic botany has become more precise and accurate. However, to develop a full proof and reproducible techniques specific to region and cases have to be developed. Using of plant based public database and its development according to the need of forensic analyst will help the investigating officer for easy and comparatively fast closure of the case. Training at various levels and multitier programmes should start at university level. High level centre for excellence is the need of hour. Allocation of adequate fund for training, research and development will help to enhance the utility and develop human resources.